PET Imaging of Neurofibromatosis Type 1 with a Fluorine-18 Labeled Tryptophan Radiotracer.

Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder. Plexiform neurofibromas (PNFs) are benign tumors commonly formed in patients with NF1. PNFs have a high incidence of developing into malignant peripheral nerve sheath tumors (MPNSTs) with a 5-year survival rate of only 30%. Therefore, the accurate diagnosis and differentiation of MPNSTs from benign PNFs are critical to patient management. We studied a fluorine-18 labeled tryptophan positron emission tomography (PET) radiotracer, 1-(2-[18F]fluoroethyl)-L-tryptophan (L-[18F]FETrp), to detect NF1-associated tumors in an animal model. An ex vivo biodistribution study of L-[18F]FETrp showed a similar tracer distribution and kinetics between the wild-type and triple mutant mice with the highest uptake in the pancreas. Bone uptake was stable. Brain uptake was low during the 90-min uptake period. Static PET imaging at 60 min post-injection showed L-[18F]FETrp had a comparable tumor uptake with [18F]fluorodeoxyglucose (FDG). However, L-[18F]FETrp showed a significantly higher tumor-to-brain ratio than FDG (n = 4, p < 0.05). Sixty-minute-long dynamic PET scans using the two radiotracers showed similar kidney, liver, and lung kinetics. A dysregulated tryptophan metabolism in NF1 mice was further confirmed using immunohistostaining. L-[18F]FETrp is warranted to further investigate differentiating malignant NF1 tumors from benign PNFs. The study may reveal the tryptophan-kynurenine pathway as a therapeutic target for treating NF1.

Clinical application of magnetic resonance elastography in pediatric neurological disorders.

Magnetic resonance elastography is a relatively new, rapidly evolving quantitative magnetic resonance imaging technique which can be used for mapping the viscoelastic mechanical properties of soft tissues. MR elastography measurements are akin to manual palpation but with the advantages of both being quantitative and being useful for regions which are not available for palpation, such as the human brain. MR elastography is noninvasive, well tolerated, and complements standard radiological and histopathological studies by providing in vivo measurements that reflect tissue microstructural integrity. While brain MR elastography studies in adults are becoming frequent, published studies on the utility of MR elastography in children are sparse. In this review, we have summarized the major scientific principles and recent clinical applications of brain MR elastography in diagnostic neuroscience and discuss avenues for impact in assessing the pediatric brain.

Special Issue "In Vivo Nuclear Molecular Imaging in Drug Development and Pharmacological Research".

Nuclear molecular imaging is increasingly important in aiding diagnosis, monitoring disease progression, and assessing response to treatment [...].

Abusive Head Trauma Animal Models: Focus on Biomarkers.

Abusive head trauma (AHT) is a serious traumatic brain injury and the leading cause of death in children younger than 2 years. The development of experimental animal models to simulate clinical AHT cases is challenging. Several animal models have been designed to mimic the pathophysiological and behavioral changes in pediatric AHT, ranging from lissencephalic rodents to gyrencephalic piglets, lambs, and non-human primates. These models can provide helpful information for AHT, but many studies utilizing them lack consistent and rigorous characterization of brain changes and have low reproducibility of the inflicted trauma. Clinical translatability of animal models is also limited due to significant structural differences between developing infant human brains and the brains of animals, and an insufficient ability to mimic the effects of long-term degenerative diseases and to model how secondary injuries impact the development of the brain in children. Nevertheless, animal models can provide clues on biochemical effectors that mediate secondary brain injury after AHT including neuroinflammation, excitotoxicity, reactive oxygen toxicity, axonal damage, and neuronal death. They also allow for investigation of the interdependency of injured neurons and analysis of the cell types involved in neuronal degeneration and malfunction. This review first focuses on the clinical challenges in diagnosing AHT and describes various biomarkers in clinical AHT cases. Then typical preclinical biomarkers such as microglia and astrocytes, reactive oxygen species, and activated N-methyl-D-aspartate receptors in AHT are described, and the value and limitations of animal models in preclinical drug discovery for AHT are discussed.

Advanced Neuroimaging Approaches to Pediatric Brain Tumors.

Central nervous system tumors are the most common pediatric solid tumors; they are also the most lethal. Unlike adults, childhood brain tumors are mostly primary in origin and differ in type, location and molecular signature. Tumor characteristics (incidence, location, and type) vary with age. Children present with a variety of symptoms, making early accurate diagnosis challenging. Neuroimaging is key in the initial diagnosis and monitoring of pediatric brain tumors. Conventional anatomic imaging approaches (computed tomography (CT) and magnetic resonance imaging (MRI)) are useful for tumor detection but have limited utility differentiating tumor types and grades. Advanced MRI techniques (diffusion-weighed imaging, diffusion tensor imaging, functional MRI, arterial spin labeling perfusion imaging, MR spectroscopy, and MR elastography) provide additional and improved structural and functional information. Combined with positron emission tomography (PET) and single-photon emission CT (SPECT), advanced techniques provide functional information on tumor metabolism and physiology through the use of radiotracer probes. Radiomics and radiogenomics offer promising insight into the prediction of tumor subtype, post-treatment response to treatment, and prognostication. In this paper, a brief review of pediatric brain cancers, by type, is provided with a comprehensive description of advanced imaging techniques including clinical applications that are currently utilized for the assessment and evaluation of pediatric brain tumors.

Radiosynthesis of 1-(2-[18F]Fluoroethyl)-L-Tryptophan using a One-pot, Two-step Protocol.

The kynurenine pathway (KP) is a primary route for tryptophan metabolism. Evidence strongly suggests that metabolites of the KP play a vital role in tumor proliferation, epilepsy, neurodegenerative diseases, and psychiatric illnesses due to their immune-modulatory, neuro-modulatory, and neurotoxic effects. The most extensively used positron emission tomography (PET) agent for mapping tryptophan metabolism, α-[11C]methyl-L-tryptophan ([11C]AMT), has a short half-life of 20 min with laborious radiosynthesis procedures. An onsite cyclotron is required to radiosynthesize [11C]AMT. Only a limited number of centers produce [11C]AMT for preclinical studies and clinical investigations. Hence, the development of an alternative imaging agent that has a longer half-life, favorable in vivo kinetics, and is easy to automate is urgently needed. The utility and value of 1-(2-[18F]fluoroethyl)-L-tryptophan, a fluorine-18-labeled tryptophan analog, has been reported in preclinical applications in cell line-derived xenografts, patient-derived xenografts, and transgenic tumor models. This paper presents a protocol for the radiosynthesis of 1-(2-[18F]fluoroethyl)-L-tryptophan using a one-pot, two-step strategy. Using this protocol, the radiotracer can be produced in a 20 ± 5% (decay corrected at the end of synthesis, n > 20) radiochemical yield, with both radiochemical purity and enantiomeric excess of over 95%. The protocol features a small precursor amount with no more than 0.5 mL of reaction solvent in each step, low loading of potentially toxic 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K222), and an environmentally benign and injectable mobile phase for purification. The protocol can be easily configured to produce 1-(2-[18F]fluoroethyl)-L-tryptophan for clinical investigation in a commercially available module.

Unravelling neuroinflammation in abusive head trauma with radiotracer imaging.

Abusive head trauma (AHT) is a leading cause of mortality and morbidity in child abuse, with a mortality rate of approximately 25%. In survivors, the prognosis remains dismal, with high prevalence of cerebral palsy, epilepsy and neuropsychiatric disorders. Early and accurate diagnosis of AHT is challenging, both clinically and radiologically, with up to one-third of cases missed on initial examination. Moreover, most of the management in AHT is supportive, reflective of the lack of clear understanding of specific pathogenic mechanisms underlying secondary insult, with approaches targeted toward decreasing intracranial hypertension and reducing cerebral metabolism, cell death and excitotoxicity. Multiple studies have elucidated the role of pro- and anti-inflammatory cytokines and chemokines with upregulation/recruitment of microglia/macrophages, oligodendrocytes and astrocytes in severe traumatic brain injury (TBI). In addition, recent studies in animal models of AHT have demonstrated significant upregulation of microglia, with a potential role of inflammatory cascade contributing to secondary insult. Despite the histological and biochemical evidence, there is a significant dearth of specific imaging approaches to identify this neuroinflammation in AHT. The primary motivation for development of such imaging approaches stems from the need to therapeutically target neuroinflammation and establish its utility in monitoring and prognostication. In the present paper, we discuss the available data suggesting the potential role of neuroinflammation in AHT and role of radiotracer imaging in aiding diagnosis and patient management.

Birth-related subdural hemorrhage: prevalence and imaging morphology.

BACKGROUND: Birth trauma accounts for 1-2% of the mortality in newborns with significant intracranial injuries presenting in the immediate postnatal period. However, a significant number of asymptomatic neonates harbor birth-related intracranial hemorrhage (ICH), with birth-related subdural hemorrhage (SDH) being a common occurrence on infant brain CT and MRI studies performed as a standard of care for a variety of reasons. Although clinically insignificant, birth-related SDH is frequently brought up in courts as an alternative explanation for SDH in suspected abusive head trauma. OBJECTIVE: The aim of this study was to determine prevalence, imaging morphology and distribution of birth-related SDHs on brain CT and MRI studies obtained as a standard of care in infants up to 1 month old. We further tried to ascertain the relationship of birth-related SDHs with mode of delivery and birth weight. MATERIALS AND METHODS: Infants up to the age of 1 month who had CT or MRI of the brain performed between Jan. 1, 2018, and March 29, 2020, were included in this retrospective observational study. In addition to the imaging data, we reviewed clinical history, birth history including birth weight and mode of delivery, and final diagnoses. RESULTS: Two hundred six infants younger than 30 days (range 0-29 days, mean 11.9 days, median 11 days and standard deviation [SD] 8.4 days) had a CT or MRI study during the study period. Among these, 58 infants were excluded as per the exclusion criteria. Among the included 148 infants, 88 (59.5%) had no imaging evidence of SDH. An additional 56 (37.8%) infants were assessed as having birth-related SDH based on review of clinical data. Within the birth-related SDH cohort (56 infants), only supratentorial SDH was identified in 5 (8.9%), only infratentorial SDH was identified in 14 (25%), while SDHs within both compartments were identified in 37 (66.1%) infants. The most common location for supratentorial birth-related SDH was along the occipital lobes (31/42, 73.8%), with other common locations being along the posterior interhemispheric fissure (30/42, 71.4%) and fronto-parietal convexity (9/42, 21.4%). The distribution of posterior fossa SDH was along the tentorium (38/51, 74.5%), along the cerebellum (38/51, 74.5%) and in both the locations (25/51, 49.0%). The rate of SDH was significantly higher in vaginal delivery group (46/84, 54.7%) as compared to caesarean section group (10/57, 17.5%) (P<0.05). We did not find any statistically significant difference between the birth weights of normal and birth-related SDH cohorts (P>0.05). CONCLUSION: Birth-related SDH is a common occurrence, with our study suggesting a prevalence of 37.8%. The most common distribution of birth-related SDH is within both the supra- and infratentorial compartments (66.1%) followed by infratentorial compartment (25%). The rate of birth-related SDH was significantly higher in vaginal delivery group as compared to caesarean section group.

PET imaging of medulloblastoma with an 18F-labeled tryptophan analogue in a transgenic mouse model.

In vivo positron emission tomography (PET) imaging is a key modality to evaluate disease status of brain tumors. In recent years, tremendous efforts have been made in developing PET imaging methods for pediatric brain tumors. Carbon-11 labelled tryptophan derivatives are feasible as PET imaging probes in brain tumor patients with activation of the kynurenine pathway, but the short half-life of carbon-11 limits its application. Using a transgenic mouse model for the sonic hedgehog (Shh) subgroup of medulloblastoma, here we evaluated the potential of the newly developed 1-(2-[18F]fluoroethyl)-L-tryptophan (1-L-[18F]FETrp) as a PET imaging probe for this common malignant pediatric brain tumor. 1-L-[18F]FETrp was synthesized on a PETCHEM automatic synthesizer with good chemical and radiochemical purities and enantiomeric excess values. Imaging was performed in tumor-bearing Smo/Smo medulloblastoma mice with constitutive actvation of the Smoothened (Smo) receptor using a PerkinElmer G4 PET-X-Ray scanner. Medulloblastoma showed significant and specific accumulation of 1-L-[18F]FETrp. 1-L-[18F]FETrp also showed significantly higher tumor uptake than its D-enantiomer, 1-D-[18F]FETrp. The uptake of 1-L-[18F]FETrp in the normal brain tissue was low, suggesting that 1-L-[18F]FETrp may prove a valuable PET imaging probe for the Shh subgroup of medulloblastoma and possibly other pediatric and adult brain tumors.

Automated production of 1-(2-[18F]fluoroethyl)-l-tryptophan for imaging of tryptophan metabolism.

Automated production of an fluorine-18 labeled tryptophan analogue, 1-(2-[18F]fluoroethyl)-l-tryptophan (1-L-[18F]FETrp) in a current Good Manufacturing Practice facility was achieved. 1-L-[18F]FETrp was produced by a one-pot, two-step strategy with an overall synthesis time of approximately 100 min, a radiochemical yield of 20 ± 5% (decay corrected), radiochemical purity and enantiomeric excess over 90%, and a molar activity of 103 ± 15 GBq/µmol at the end of synthesis (EOS). The dose mass of 1-L-FETrp in four consecutive batches was less than 5 µg. The radiopharmaceutical product met all quality control criteria for clinical use.

Enzymatic activity of palmitoyl-protein thioesterase-1 in serum from schizophrenia significantly associates with schizophrenia diagnosis scales.

Genome-wide association studies have confirmed that schizophrenia is an inheritable multiple-gene mental disorder. Longitudinal studies about depression, first episode psychosis (FEP) and acute psychotic relapse have mostly searched for brain imaging biomarkers and inflammatory markers from the blood. However, to the best of our knowledge, the association between enzymatic activities with diagnosis or prediction of treatment response in people with schizophrenia has barely been validated. Under the Longitudinal Study of National Mental Health Work Plan (2015-2020), we have studied a subsample of approximately 36 individuals from the cohort with data on palmitoyl-protein thioesterase-1 enzymatic activity from FEP and performed a bivariate correlation analysis with psychiatric assessment scores. After adjusting for sex, age, body mass index (BMI) and total serum protein, our data demonstrated that PPT1 enzymatic activity is significantly associated with schizophrenia and its Positive and Negative Syndrome Scale (PANSS) scores. This longitudinal study compared the PPT1 enzymatic activity in FEP schizophrenia patients and healthy volunteers, and the former exhibited a significant 1.5-fold increase in PPT1 enzymatic levels (1.79 mmol/L/h/mL, and 1.18 mmol/L/h/mL; P < 0.05; 95% CI, 2.3-2.9 and 1.4-1.8). The higher PPT1 enzymatic levels in FEP schizophrenia patients were positively associated with larger PANSS scaling scores (r = 0.32, P = 0.0079 for positive scaling; r = 0.41, P = 0.0006 for negative scaling; r = 0.45, P = 0.0001 for general scaling; and r = 0.34, P = 0.0048 for PNASS-S scaling). Higher enzymatic PPT1 in FEP schizophrenia patients is significantly associated with increased PANSS scaling values, indicating more serious rates of developing psychosis. Enzymatic activity of PPT1 may provide an important new view for schizophrenia disorders.

Automated production of a N-methyl-D-aspartate receptor radioligand [18F]GE179 for clinical use.

N-Methyl-d-aspartate (NMDA) receptors are ligand and voltage-gated heteromeric ion channel receptors. Excessive activation of NMDA receptors is implicated in many neurological and psychiatric disorders, including ischemic stroke, neuropathic pain, epilepsy, drug addition, Alzheimer's disease, and schizophrenia. [18F]GE179 is a promising PET probe for imaging functional NMDA receptor alterations (activated or 'open' channel) with a high binding affinity (Kd = 2.4 nM). Here, we report the production of the NMDA receptor radioligand [18F]GE179 in a current Good Manufacturing Practice (cGMP) facility through a one-pot two-step strategy. [18F]GE179 was produced in approximately 110 min with a radiochemical yield of 12 ±â€¯6% (n = 4, decay corrected), radiochemical purity >95%, molar activity of 146 ±â€¯32 GBq/µmol (at the end of synthesis), an average mass of GE179 at 2.2 µg/batch, and total impurities less than 0.5 µg/batch (n = 4). The radiopharmaceutical dose meets all quality control (QC) criteria for human use, and is suitable for clinical PET studies of activated NMDA receptor ion channels.

Radiosynthesis and evaluation of a fluorine-18 labeled radioligand targeting vesicular acetylcholine transporter.

Vesicular acetylcholine transporter (VAChT) is a reliable biomarker for assessing the loss of cholinergic neurons in the brain that is associated with cognitive impairment of patients. 5-Hydrotetralin compound (±)-5-OH-VAT is potent (Ki = 4.64 ±â€¯0.32 nM) and selective for VAChT (>1800-fold and 398-fold for σ1 and σ2 receptor, respectively) with favorable hydrophilicity (LogD = 1.78), while (-)-5-OH-VAT originally serves as the radiolabeling precursor of (-)-[18F]VAT, a promising VAChT radiotracer with a logD value of 2.56. To evaluate (-)-5-OH-[18F]VAT as a radiotracer for VAChT, we performed in vitro binding assay to determine the potency of the minus enantiomer (-)-5-OH-VAT and plus enantiomer (+)-5-OH-VAT, indicating that (-)-5-OH-VAT is a more potent VAChT enantiomer. Radiosynthesis of (-)-5-OH-[18F]VAT was explored using three strategies. (-)-5-OH-[18F]VAT was achieved with a good yield (24 ±â€¯6%) and high molar activity (∼37 GBq/µmol, at the end of synthesis) using a microwave assisted two-step one-pot procedure that started with di-MOM protected nitro-containing precursor (-)-6. MicroPET studies in the brain of nonhuman primate (NHP) suggest that (-)-5-OH-[18F]VAT readily penetrated the blood brain barrier and specifically accumulated in the VAChT-enriched striatum with improved washout kinetics from striatum compared to [18F]VAT. Nevertheless, the lower target to non-target ratio may limit its use for in vivo measurement of the VAChT level in the brain.

Design, synthesis, and in vitro evaluation of quinolinyl analogues for α-synuclein aggregation.

Here we report the synthesis and in vitro evaluation of 25 new quinolinyl analogues for α-synuclein aggregates. Three lead compounds were subsequently labeled with carbon-11 or fluorine-18 to directly assess their potency in a direct radioactive competitive binding assay ng both α-synuclein fibrils and tissue homogenates from Alzheimer's disease (AD) cases. The modest binding affinities of these three radioligands toward α-synuclein were comparable with results from the Thioflavin T fluorescence assay. However, all three ligand also showed modest binding affinity to the AD homogenates and lack selectivity for α-synuclein. The structure-activity relationship data from these 25 analogues will provide useful information for design and synthesis of new compounds for imaging α-synuclein aggregation.

In Vivo Characterization of Two 18F-Labeled PDE10A PET Radioligands in Nonhuman Primate Brains.

Positron emission tomography (PET) with phosphodiesterase 10A (PDE10A) specific radioligands provides a noninvasive and quantitative imaging tool to access the expression of this enzyme in vivo under normal and diseased conditions. We recently reported two potent 18F-labeled PDE10A radioligands (18F-TZ19106B and 18F-TZ8110); initial evaluation in rats and nonhuman primates indicated stable metabolic profiles and excellent target-to-nontarget ratio (striatum/cerebellum) for both tracers. Herein, we focused on in vivo characterization of 18F-TZ19106B and 18F-TZ8110 to identify a suitable radioligand for imaging PDE10A in vivo. We directly compared microPET studies of these two radiotracers in adult male Macaca fascicularis nonhuman primates (NHPs). 18F-TZ19106B had higher striatal uptake and tracer retention in NHP brains than 18F-TZ8110, quantified by either standardized uptake values (SUVs) or nondisplaceable binding potential (BPND) estimated using reference-based modeling analysis. Blocking and displacement studies using the PDE10A inhibitor MP-10 indicated the binding of 18F-TZ19106B to PDE10A was specific and reversible. We also demonstrated sensitivity of 18F-TZ19106B binding to varying number of specific binding sites using escalating doses of MP-10 blockade (0.3, 0.5, 1.0, 1.5, and 2.0 mg/kg). Pretreatment with a dopamine D2-like receptor antagonist enhanced the striatal uptake of 18F-TZ19106B. Our results indicate that 18F-TZ19106B is a promising radioligand candidate for imaging PDE10A in vivo and it may be used to determine target engagement of PDE10A inhibitors and serve as a tool to evaluate the effect of novel antipsychotic therapies.

Cholinergic imbalance in lumbar spinal cord of a rat model of multiple sclerosis.

Cholinergic dysfunction in the central nervous system is an important characteristic of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). By using a rat EAE model, upregulation of vesicular acetylcholine transporter (VAChT) level in the EAE rat lumbar spinal cord was detected by western blot and immunostaining, and was associated with lymphocyte filtration and glial activation. Ex vivo and in vitro autoradiography studies with [18F]VAT, a VAChT-specific radioligand, also revealed increased tracer uptake in EAE rat lumbar spinal cord compared with shams. These studies on VAChT expression suggest central cholinergic imbalance during EAE progression.

Kinetic modeling of [18 F]VAT, a novel radioligand for positron emission tomography imaging vesicular acetylcholine transporter in non-human primate brain.

Molecular imaging of vesicular acetylcholine transporter (VAChT) in the brain provides an important cholinergic biomarker for the pathophysiology and treatment of dementias including Alzheimer's disease. In this study, kinetics modeling methods were applied and compared for quantifying regional brain uptake of the VAChT-specific positron emission tomography radiotracer, ((-)-(1-(-8-(2-fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(4-fluorophenyl)-methanone) ([18 F]VAT) in macaques. Total volume distribution (VT ) estimates were compared for one-tissue compartment model (1TCM), two-tissue compartment model (2TCM), Logan graphic analysis (LoganAIF) and multiple linear analysis (MA1) with arterial blood input function using data from three macaques. Using the cerebellum-hemispheres as the reference region with data from seven macaques, three additional models were compared: reference tissue model (RTM), simplified RTM (SRTM), and Logan graphic analysis (LoganREF). Model selection criterion indicated that a) 2TCM and SRTM were the most appropriate kinetics models for [18 F]VAT; and b) SRTM was strongly correlated with 2TCM (Pearson's coefficients r > 0.93, p < 0.05). Test-retest studies demonstrated that [18 F]VAT has good reproducibility and reliability (TRV < 10%, ICC > 0.72). These studies demonstrate [18 F]VAT is a promising VAChT positron emission tomography tracer for quantitative assessment of VAChT levels in the brain of living subjects.

Synthesis and in vitro characterization of a P2X7 radioligand [123I]TZ6019 and its response to neuroinflammation in a mouse model of Alzheimer disease.

The purinergic receptor P2X ligand-gated ion channel 7 (P2X7 receptor) is a promising imaging target to detect neuroinflammation. Herein, we report development of a potent iodinated radiotracer for P2X7 receptor, [123I]TZ6019. The radiosynthesis of [123I]TZ6019 was accomplished by allylic-tin precursor iodination using [123I]NaI with good radiochemical yield of 85% and high radiochemical purity of > 99%. Human embryonic kidney 293 (HEK-293) cell line stably transfected with the human P2X7 receptor was used to characterize the binding affinity of TZ6019 by fluorescence, radioactive competitive, and saturation binding assays. A radioligand competitive binding assay with [123I]TZ6019 demonstrated that the nonradioactive compound TZ6019 has an IC50 value of 9.49 ± 1.4nM, and the known P2X7 receptor compound GSK1482160 has an IC50 value of 4.30 ± 0.86nM, consistent with previous reports. The radioligand saturation binding assay and competitive assay revealed that [123I]TZ6019 specifically bound to the human P2X7 receptor with high affinity (Ki = 6.3 ± 0.9nM). In vitro autoradiography quantification with brain slices collected from 9-month old P301S tau transgenic mice along with wild type controls, revealed higher binding of [123I]TZ6019 (35% increase) in the brain of P301S transgenic mice (n = 3, p = 0.04) compared to wild type controls. The immunofluorescence microscopy confirmed that expression of P2X7 receptor was colocalized with astrocytes in the tauopathy P301S transgenic mice. [123I]TZ6019 has specific binding for P2X7 receptor and has great potential to be a radiotracer for screening new compounds and quantifying expression of P2X7 receptor in neuroinflammation related diseases.

Design and synthesis of pyrazolopyridine derivatives as sphingosine 1-phosphate receptor 2 ligands.

Eleven new sphingosine 1-phosphate receptor 2 (S1PR2) ligands were synthesized by modifying lead compound N-(2,6-dichloropyridin-4-yl)-2-(4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridin-6-yl)hydrazine-1-carboxamide (JTE-013) and their binding affinities toward S1PRs were determined in vitro using [32P]S1P and cell membranes expressing recombinant human S1PRs. Among these ligands, 35a (IC50 = 29.1 ±â€¯2.6 nM) and 35b (IC50 = 56.5 ±â€¯4.0 nM) exhibit binding potency toward S1PR2 comparable to JTE-013 (IC50 = 58.4 ±â€¯7.4 nM) with good selectivity for S1PR2 over the other S1PRs (IC50 > 1000 nM). Further optimization of these analogues may identify additional and more potent and selective compounds targeting S1PR2.

Upregulated Sphingosine 1-Phosphate Receptor 1 Expression in Human and Murine Atherosclerotic Plaques.

PURPOSE: Dysregulation of sphingosine 1-phosphate receptor 1 (S1PR1) signaling contributes to inflammation-related pathophysiological changes in cardiovascular diseases including atherosclerosis (AS). S1PR1-targeting compounds significantly reduce lesion size in murine models of AS. Therefore, characterization of S1PR1 expression in vitro and in vivo in atherosclerotic plaque could enable mechanistic studies and inform S1PR1 targeted therapies. PROCEDURES: H&E staining and immunostaining studies were performed on variably diseased human femoral endarterectomy plaque specimens, as well as mouse aortic sections from ApoE-/- mice maintained on a high-fat diet (AS mice). In vitro autoradiography study in human femoral plaques was used to confirm the tracer specificity. Micro positron emission tomography (PET) and ex vivo autoradiography studies were conducted in AS mice and their controls using a S1PR1-specific radioligand [11C]TZ3321 for in vivo and ex vivo quantification of S1PR1 expression in mouse aortic plaques. RESULTS: Increased S1PR1 expression was observed in areas of human femoral endarterectomy plaque specimens with foam cell accumulation compared with control tissue; in vitro autoradiography study indicated that SEW2781, a S1PR1 compound was able to reduce the uptake of [11C]TZ3321 by 56 %. S1PR1 levels were also upregulated in AS mouse aortic plaques. MicroPET data showed the aorta-to-blood tracer uptake ratio in AS mice was approximately 20 % higher than that in controls. Autoradiographic study also revealed elevated tracer accumulation in AS mouse aorta. CONCLUSIONS: Upregulated S1PR1 expression in human and mouse atherosclerotic plaques was successfully identified by immunostaining and radioligand-based methods. This data demonstrates that [11C]TZ3321 PET provides great promise in imaging S1PR1 expression in atherosclerotic plaques.